CD22 is a B lymphocyte specific glycan binding protein that participates in regulation of B cell receptor signaling. The extracellular domain recognizes sialic acid containing glycans as ligands, which are abundantly expressed as glycan chains of glycoproteins on B lymphocytes, T lymphocytes, and other cells that B lymphocytes come in contact with during activation and differentiation. In this project we seek to understand how CD22, regulates the activation and differentiation of B lymphocytes, and in particular to understand the impact of glycoprotein ligands on CD22 function. The major objectives of the project are to: 1) Identify the ligands of CD22 on B cells and T cells. This will be accomplished through use of a method we developed for crosslinking glycan ligands to CD22. CD22 can then be used as a tag to fish out the glycoprotein ligands, and identify them through standard proteomics and immunological methods. 2) Assess the roles of CD22 ligands in the B cell immune response. This will be done through the creation of transgenic mice devoid of CD22 ligands (ST6Gal I null) that produce antigen specific B cells and T cells. Purified B cells and T cells from these mice can be delivered to wild type mice to assess the role of CD22 ligands in response to antigen mediated activation and differentiation of B cells in a natural wild type environment. 3) Evaluate the roles of cytoplasmic motifs of CD22 in ligand binding, microdomain localization, endocytosis and recruitment of signaling cofactors. We hope to identify mutants that are deficient in either endocytosis or cofactor recruitment, in order to study the role of CD22 endocytosis in BCR signaling. 4) Create transgenic mice with CD22 mutants selectively deficient in recruitment of SHP-1 and/or endocytosis to assess their distinct roles in CD22 regulation of B lymphocyte signaling in vivo. The results of objective 3 will lead to the creation of transgenic mice that can be used to confirm in vitro findings in an in vivo setting. Public Health Relevance Statement: The general significance of this project is directed to a deeper understanding of basic processes that regulate immune defense by lymphocytes. Lymphocytes are the major mediators of adaptive immunity, responding to antigens, pathogens and other non-self substances, in a highly specific manner. Immunization of an individual with antigen containing vaccines programs lymphocytes to rapidly respond to a disease agent and prevent infection. Aberrant activation of lymphocytes leads to chronic inflammatory disease such as lupus, rheumatoid arthritis and other diseases. Lymphocytes can also recognize some differences between normal cells and cancer cells, and destroy the cancer cell as non-self. Thus, a deeper understanding of the basic immune processes studied in this project will aid in the development of novel therapeutic approaches infectious disease, chronic inflammatory disease and cancer.